Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37°C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28°C by all cathepsins but cathepsin K, whereas thermal destabilization at 37°C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.Controlled degradation of collagen is observed in bone remodeling, wound healing, angiogenesis, and during organ development (1-3). On the other hand excessive collagen degradation leads to pathological phenotypes such as osteoporosis (4), various forms of arthritis (5), or aneurysms of blood vessels (6), or it is characteristic for tumor invasion (7). However, triple helical collagens, in particular type I and II collagens, are highly resistant to general proteolysis and require specific proteases for their degradation. Known mammalian collagenolytic activities include members of the matrix metalloprotease family such as MMP-1, -2, -8, -13, and -14 (8), the serine protease, human neutrophil elastase (9), and thiol-dependent cathepsins (1). Collagenases of the MMP family cleave triple helical collagen at a specific single site and release 3 ⁄4 and 1 ⁄4 fragments. Similar to MMPs, human neutrophil elastase generates 3 ⁄4 fragments from type I collagen but is unable to degrade type II collagen (9). Lysosomal cysteine proteases such as cathepsins L and B have also been discussed as collagenolytic activities, but these data were mostly based on inhibitor experiments in cell extracts or on early preparations of cathepsins, which may not have excluded contaminating activities (10 -13). Thorough enzymatic studies suggested that cathepsins B and L primarily cleave in the non-helical telopeptide extensions of collagens (14, 15). A truly triple helical collagenase activity is found in cathepsin K, which is predominantly expressed in osteoclasts and to a lower degree in various other cell types including fibroblasts (16 -19). It was demonstrated that cathepsin K, similar to the bacterial Clostridium collagenase, cleaves at multiple sites within the triple helical region of types I and II collagens (20,21). The biological relevan...

Cathepsin V, a thymus and testis-specific human cysteine protease, was expressed in Pichia pastoris, and its physicokinetic properties were determined. Recombinant procathepsin V is autocatalytically activated at acidic pH and is effectively inhibited by various cysteine protease class-specific inhibitors. The S2P2 subsite specificity of cathepsin V was found to be intermediate between those of cathepsins S and L. The substrate binding pocket, S2, accepted both aromatic and nonaromatic hydrophobic residues, whereas cathepsins L and S preferred either an aromatic or nonaromatic hydrophobic residue, respectively. In contrast to cathepsin L, but similar to cathepsin S, cathepsin V exhibited only a very weak collagenolytic activity. Furthermore, cathepsin V was determined to be significantly more stable at mildly acidic and neutral pH than cathepsin L, but distinctly less stable than cathepsin S. A homology structure model of cathepsin V revealed completely different electrostatic potentials on the molecular surface when compared with human cathepsin L. The model-based electrostatic potential of human cathepsin V was neutral to weakly positive at and in the vicinity of the active site cleft, whereas that of cathepsin L was negative over extended regions of the surface. Surprisingly, the electrostatic potential of the human cathepsin V model structure resembled that of the model structure of mouse cathepsin L. These differences in the electrostatic potential at the molecular surfaces provide a reactivity determinant that may be the source of differences in substrate selectivity and pH stability. Cathepsin V was mapped to the chromosomal region 9q22.2, a site adjacent to the cathepsin L locus. The high sequence identity and the overlapping chromosomal gene loci suggest that both proteases evolved from an ancestral cathepsin L-like precursor by gene duplication.

The major histocompatibility complex (MHC) class II–associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a ∼3-kD peptide termed CLIP (class II–associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii–MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

Bone resorption in balance with bone formation is vital for the maintenance of the skeleton and is mediated by osteoclasts. Cathepsin K is the predominant protease in osteoclasts that degrades the bulk of the major bone forming organic component, type I collagen. Although the potent collagenase activity of cathepsin K is well known, its mechanism of action remains elusive. Here, we report a cathepsin K-specific complex with chondroitin sulfate, which is essential for the collagenolytic activity of the enzyme. The complex is an oligomer consisting of five cathepsin K and five chondroitin sulfate molecules. Only the complex exhibits potent triple helical collagen-degrading activity, whereas monomeric cathepsin K has no collagenase activity. The primary substrate specificity of cathepsin K is not altered by complex formation, suggesting that the protease-chondroitin sulfate complex primarily facilitates the destabilization and/or the specific binding of the triple helical collagen structure. Inhibition of complex formation leads to the loss of collagenolytic activity but does not impair the proteolytic activity of cathepsin K toward noncollagenous substrates. The physiological relevance of cathepsin K complexes is supported by the findings that (i) the content of chondroitin sulfate present in bone and accessible to cathepsin K activity is sufficient for complex formation and (ii) Y212C, a cathepsin K mutant that causes pycnodysostosis (a bone sclerosing disorder) and that has no collagenase activity but remains potent as a gelatinase, is unable to form complexes. These findings reveal a novel mechanism of bone collagen degradation and suggest that targeting cathepsin K complex formation would be an effective and specific treatment for diseases with excessive bone resorption such as osteoporosis.

Synovial fibroblasts (SFs) play a critical role in the pathogenesis of rheumatoid arthritis (RA) and are directly involved in joint destruction. Both SF-resident matrix metalloproteases and cathepsins have been implicated in cartilage degradation although their identities and individual contributions remain unclear. The aims of this study were to investigate the expression of cathepsin K in SFs, the correlation between cathepsin K expression and disease severity, and the contribution of cathepsin K to fibroblast-mediated collagen degradation. Immunostaining of joint specimens of 21 patients revealed high expression of cathepsin K in SFs in the synovial lining and the stroma of synovial villi, and to a lesser extent in CD68-positive cells of the synovial lining. Cathepsin K-positive SFs were consistently observed at sites of cartilage and bone degradation. Expression levels of cathepsin K in the sublining and vascularized areas of inflamed synovia showed a highly significant negative correlation with results derived from the Hannover Functional Capacity Questionnaire (r = 0.78, P = 0.003; and r = 0.70, P = 0.012, respectively) as a measure of the severity of RA in individual patients. For comparison, there was no correlation between Hannover Functional Capacity Questionnaire and cathepsin S whose expression is limited to CD-68-positive macrophage-like synoviocytes. The expression of cathepsin K was also demonstrated in primary cell cultures of RA-SFs. Co-cultures of SFs on cartilage disks revealed the ability of fibroblast-like cells to phagocytose collagen fibrils whose intralysosomal hydrolysis was prevented in the presence of a potent cathepsin K inhibitor but not by an inhibitor effective against cathepsins L, B, and S. The selective and critical role of cathepsin K in articular cartilage and subchondral bone erosion was further corroborated by the finding that cathepsin K has a potent aggrecan-degrading activity and that cathepsin K-generated aggrecan cleavage products specifically potentiate the collagenolytic activity of cathepsin K toward type I and II collagens. This study demonstrates for the first time a critical role of cathepsin K in cartilage degradation by SFs in RA that is comparable to its well-known activity in osteoclasts.

Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.Atherosclerosis is characterized by arterial intimal enlargement and subsequent lipid deposition leading to the formation of blood stream obstructing plaques. The infiltration of monocyte-derived macrophages (MDMs) 1 and smooth muscle cells (SMCs) into the intima of the inflamed artery contributes to the formation of atherosclerotic lesions. Atherosclerotic lesions resident MDMs and SMCs produce a large number of extracellular matrix-degrading enzymes (1), such as cysteine proteases (2-5) and matrix metalloproteinases (MMPs) (6 -8).Elastin and collagen are the two major extracellular matrix components that provide elasticity and tensile strength to the arterial wall. The destruction of elastin and collagen causes a weakening and rupture of blood vessels (9, 10). MMPs, serine, and cysteine proteases have been identified as major elastolytic proteases in arteries (11, 12). Among cysteine proteases, cathepsins S and K have been considered as the most potent elastolytic activities with cathepsin K exhibiting a slightly higher activity than cathepsin S (13, 14). However, cathepsin K-deficient human macrophages derived from patients with pycnodysostosis were shown to retain their high cysteine proteasedependent elastolytic activities (15). This finding implied that additional cathepsins may contribute to the overall elastolytic activity in human macrophages.We and others (16 -18) have identified and partially characterized a novel human cysteine protease closely related to cathepsin L that was named cathepsin V (also known as cathepsin L2). Cathepsin V shares 80% protein s...

Cathepsin K is the predominant cysteine protease in osteoclast-mediated bone remodeling, and the protease is thought to be involved in the pathogenesis of diseases with excessive bone and cartilage resorption. Osteoclastic matrix degradation occurs in the extracellular resorption lacuna and upon phagocytosis within the cell's lysosomal-endosomal compartment. Since glycosaminoglycans (GAGs) are abundant in extracellular matrixes of cartilage and growing bone, we have analyzed the effect of GAGs on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. GAGs, in particular chondroitin sulfates, specifically and selectively increased the stability of cathepsin K but had no effect on cathepsin L and MMP-1. GAGs strongly enhanced the stability and, to a lesser extent, the catalytic activity of cathepsin K. To combine the activity and stability parameters, we defined a novel kinetic term, named cumulative activity (CA), which reflects the total substrate turnover during the life span of the enzyme. In the presence of chondroitin-4-sulfate (C-4S), the CA value increased 200-fold for cathepsin K but only 25-fold with chondroitin-6-sulfate (C-6S). C-4S dramatically increased the hydrolysis of soluble as well insoluble type I and II collagens, whereas the effects of C-6S and hyaluronic acid were less pronounced. C-4S acts in a concentration-dependent manner but reaches saturation at approximately 0.1%, a concentration similar to that found in the synovial fluid of arthritis patients. C-4S increased the cathepsin K-mediated release of hydroxyproline from insoluble type I collagen 10-fold but had only a less than 2-fold enhancing effect on the hydrolysis of intact cartilage. The relatively small increase in the hydrolysis of cartilage by C-4S was attributed to the endogenous chondroitin sulfate content present in the cartilage. Although C-4S increased the pH stability at neutral pH, a significant increase in the collagenolytic activity of cathepsin K at this pH was not observed, thus suggesting that the unique collagenolytic activity of cathepsin K at acidic pH is mechanistically determined and not by the enzyme's instability at neutral pH. The selective and significant stabilization and activation of cathepsin K activity by C-4S may provide a rationale for a novel mechanism to regulate the enzyme's activity during bone growth and aging, two processes known for significant changes in the GAG content.

We introduce Ego4D, a massive-scale egocentric video dataset and benchmark suite. It offers 3,670 hours of dailylife activity video spanning hundreds of scenarios (household, outdoor, workplace, leisure, etc.) captured by 931 unique camera wearers from 74 worldwide locations and 9 different countries. The approach to collection is designed to uphold rigorous privacy and ethics standards, with consenting participants and robust de-identification procedures where relevant. Ego4D dramatically expands the volume of diverse egocentric video footage publicly available to the research community. Portions of the video are accompanied by audio, 3D meshes of the environment, eye gaze, stereo, and/or synchronized videos from multiple egocentric cameras at the same event. Furthermore, we present a host of new benchmark challenges centered around understanding the first-person visual experience in the past (querying an episodic memory), present (analyzing hand-object manipulation, audio-visual conversation, and social interactions), and future (forecasting activities). By publicly sharing this massive annotated dataset and benchmark suite, we aim to push the frontier of first-person perception.